The chemiluminescent oxidation of luminol catalyzed by peroxidase finds wide employment in analytical test of antigens, antibodies and nucleic acids, and in particular blotting tests, e.g. Dot Blots, Western Blots (proteins), Southern and Northern Blots (nucleic acids).
It is known that the chemiluminescent oxidation of luminol catalyzed by peroxidase can be made faster and more efficient by adding an electron mediator, or enhancer, as shown, for example, by L. J. Kricka in Clinical Chemistry 1991; 37:1472-1481; or by L. J. Kricka, J. C: Voyta and I. Bronstein in “Chemiluminescent Methods for Detecting and Quantitating Enzyme Activity”, Methods Enzymol. 2000; 305:370-390. Several compounds have been used as electron mediators, including luciferin, 6-hydroxybenzotriazols, p-iodophenols, p-coumaric acid are described by G. H. G. Thorpe and L. J. Kricka, Methods Enzymol. 1986; 133:331; aromatic amines in U.S. Pat. No. 4,279,950; acetanilides in Eur. Pat. Appl. No. 603953 (1994); N-substituted phenothiazines in U.S. Pat. No. 5,171,688; boronic acids in U.S. Pat. No. 5,629,168. It is believed that, in the presence of an electron mediator, the oxidation of luminal catalyzed by peroxidase proceeds according to the following scheme:HRP+H2O2→HRP-I  (1)HRP-I+LH−→HRP-II+L.−  (2)HRP-II+LH−→HRP+L.−  (3)HRP-I+E→HRP-II+E.−  (4)HRP-II+E→HRP+E.−  (5)E.−+LH−→E+L.−  (6)L.−→L+LH−  (7)L+H2O2→LO22−  (8)LO22−→AP2−*  (9)AP2−*→AP2−+hν  (10)
where HRP, HRP-I and HRP-II indicate the enzyme peroxidase in the native and its two oxidized forms, respectively; LH—, L.−, L and LO22− represent luminol anion, luminol radical anion, diazaquinone and luminol peroxide; E and E.− represent the electron mediator, or primary enhancer, and its corresponding radical; finally AP2− indicates the dianion of 3-aminophthalic acid and AP2−* its excited state. According to this scheme, peroxidase HRP is oxidized by peroxide to HRP-I. The luminol anion and the primary enhancer are oxidized by HRP-I to their respective radicals with conversion of the enzyme to its HRP-II form. In turn, HRP-II oxidizes another molecule of luminol anion or of primary enhancer to their respective radicals, simultaneously regenerating the native form of the HRP enzyme, which can participate in another oxidation cycle.
The increase in light output observed in the presence of an electron mediator E is attributed to the faster generation of the key intermediate L.− (see, e.g. S. B: Vlasenko, A. A. Arefeyev, A. D. Klimov, B. B. Kim, E. L. Gorovits, A. P. Osipov, E. M. Gavrilova, A. M. Yegorov, J. Biolumin. Chemilumin. 1989; 4: 164-176). Once formed, L.− quickly dismutates to luminol anion LH− and diazaquinone, L. The diazaquinone is susceptible to nucleophilic attack on the carbonilic carbon (C═O) by peroxide, with formation of luminol peroxide. Finally, the luminol peroxide collapses to 3-aminophthalate in its excited form, AP2−* with simultaneous expulsion of molecular nitrogen. The excited state of 3-aminophthalate then emits a blue photon at 425 nm.
A further, significant increase in chemiluminescent light emission was obtained through the use of certain acylation catalysts, as described in US Pat. Appl. 2008/0241686 and in E. Marzocchi, S. Grilli, L. Della Ciana, L. Prodi, M. Mirasoli, A. Roda, Anal. Biochem. 2008; 277:189-194. These compounds, belonging to the class of 4-aminopyridines, provide a further enhancement in light output only when used in conjunction with primary, electron transfer type, enhancers. Thus, they can been described as “secondary enhancers”, see, e.g., the following references: M. M. Vdovenko, L. Della Ciana, I. Yu. Sakharov, Anal. Biochem. 2009; 392:54-58.
While the 4-aminopyridine catalysts of US Pat. Appl. 2008/0241686 have a powerful effect on the efficiency of the luminol chemiluminescent reaction, they are difficult to regulate. Even very small amounts of the compounds produce a large increase in signal. In addition, the signal decays much faster than in their absence. There are however advantages in providing formulations capable of producing an increased light output combined with a relatively low rate of signal decay. These features are especially valuable when repeated readings are necessary.
For this reasons, it is a valuable advance in the state of the art to provide secondary enhancers with a better degree of regulation of signal amplification, compared to that obtained with the 4-aminopyridines of US Pat. Appl. 2008/0241686.